A. Field of the Invention
This application deals generally with an apparatus and methods for the preparation of large DNA fragments in agarose plugs.
B. Description of the Related Art
Methods for preparing large DNA fragments in agarose are well known to those of skill in the field of molecular biology. With care, conventional methods allow molecules as large as 500 kD ("Kilo Daltons") to be prepared. However, the common steps in handling DNA, that is, pipetting and phenol extraction, introduce shear forces that reduce the length of DNA. Therefore, mechanical breakage due to handling procedures is a major hindrance to the preparation of large DNA. Once prepared, large DNA requires gentle handling but is relatively stable, particularly if the DNA is maintained at high concentrations.
To prepare DNA larger than 500 kb, it is necessary to protect the molecules from both mechanical breakage and nucleolytic degradation during the entire isolation process. Schwartz and Cantor prepared chromosomes from yeast by embedding the cells in agarose prior to solubilization and enzymatic digestion of the non-DNA components. The same general procedures originally developed for yeast chromosomes have been adapted for preparing large DNA from all other sources. In general, the typical steps in DNA preparation in this manner are as follows.
Cells to be embedded in agarose for DNA isolation can come from either unicellular organisms, multicellular tissues, or blood. Prior to being embedded in agarose, single cell suspensions must be obtained. For example, tissues must be dissociated, white blood cells must be concentrated, and cultured unicellular organisms must be harvested. The single cell suspensions are usually washed, and then, since the final DNA concentration can be critical, the cell number must be determined prior to embedding. Individual cells are embedded in agarose, which protects the DNA against breakage while allowing the free flow of solutions necessary for lysis and digestion. The samples are typically maintained in detergent and high concentrations of EDTA to inhibit nuclease activity. Proteinase K is fully active in such solutions (as are some other proteases, such as pronase) and therefore will digest cellular protein under conditions during which any endogenous enzymes are unable to modify or degrade the DNA. Material released by this digestion diffuses out of the agarose during the washes while the DNA remains trapped. Because cellular enzymes are removed completely by this procedure, samples prepared in this way are extremely stable and show little degradation during storage periods as long as several years.
Organisms that contain a cell wall often require an additional step specifically designed to disrupt the wall prior to proteinase K digestion to allow the lysis solution access to the membrane. This procedure will allow the lysis solution access to the membrane. This procedure usually will be another enzymatic step, for example, digestion of fungal cell walls to form spheroplasts, although in other cases mechanical force can be used, as for vascular plants.
DNA prepared in agarose remains available as a substrate for all the common enzymatic steps of molecular biology. Restriction enzymes recognize and cleave their target sites in embedded DNA. Additionally, other endo- and exonucleases can be used for labeling and ligation. If enzymatic steps are required for DNA prepared in agarose, it is essential to use agarose that is free of material that would inhibit these enzymes. The relative purity of different lots of agarose varies, as does the sensitivity of different enzymes to the contaminating compounds. Users should either pretest lots of agarose for compatibility with in-gel uses (using the intended enzymes) or use a commercially available agarose certified for its suitability for this purpose (e.g., InCert Agarose, FMC BioProducts). Although such agarose is expensive on a per gram basis, only small amounts are used in normal plug preparations. These expensive types of agarose are not necessary or helpful when preparing samples such as yeast chromosomes for size markers when no restriction digestion is required.
Once DNA is prepared in agarose the concentration cannot be changed. DNA samples can be prepared either in solid agarose, which then can be transferred as individual blocks, or in agarose microbeads, which can be pipetted. Each technique permits different amounts of the sample to be digested or run on a gel. Some prefer to prepare samples in solid agarose blocks, finding it more amenable to the simultaneous production of multiple samples and the exchanging of buffers prior to digestion. For procedures that are extremely sensitive to residual reagents, the greater surface area of microbeads permits more extensive dialysis, although loss of DNA from the surfaces of the beads lowers the yield.
As agarose embedment has become more accepted in the art, various apparatus for molding agarose plugs have been developed. Early sample molds allowed formation of one plug at a time, which was tedious and required different molds to prepare samples of different thicknesses. Two alternative molds for preparing solid samples in batches having long shallow channels (whose width and depth correspond to the dimensions of the sample comb) milled in acrylic blocks were then developed. The open side of each groove is covered with tape, and agarose-DNA mixture is pipetted in from one end. After the agarose solidifies, the long ribbon of sample is cut to convenient lengths before being subject to enzymatic treatment.
It is possible to prepare samples in square acrylic tubing by taping one end closed prior to pipetting in the agarose-DNA mixture. The tube can be placed in ice for rapid hardening; the long square "noodle" slips out of the tube easily after removal of the tape. Samples can be digested and washed intact. Samples similarly can be formed in TYGON tubing, 1-ml syringes with the front end cut off, plastic drinking straws, or glass pipets. Using round samples results in slightly more DNA migrating in the center of each lane than near the edges. Individual samples of constant thickness are sliced from the ends using a template. Major benefits of this "noodle" method are that uniformly sized samples can be prepared rapidly with large volumes of sample and, by using slicing templates with gaps of different widths, samples can be sliced accurately to different thicknesses. This variability permits different amounts of DNA to be handled in blocks of similar height and width for greater uniformity in digestion and electrophoresis.
Unfortunately, previously described devices have required that plugs be cast, and then processed in a separate device. These methods for preparing and embedding cells in agarose involved the movement of the individual plugs from solution to solution, this occurs about ten times during the development of each plug. Agarose plugs are very fragile, and breakage can occur during the transfer process. Given the fragile nature of large DNA molecules and the agarose plugs in which they are prepared, a device that would allow cells to be embedded in agarose and then processed in the same device would greatly reduce the manipulation and therefore degradation of large DNA molecules.